A. Field of the Invention
The present invention is related to the recombinant expression of proteins from eukaryotic cells. More particularly, the invention relates to the production of recombinant proteins from genetically engineered secretory cells. Methods for use of the cells also are provided.
B. Related Art
Mammalian cells of neuroendocrine origin have been used extensively over the last fifteen years as systems for the study of pathways and mechanisms of polypeptide secretion (Burgess and Kelly, 1987 and Chavez et al., 1994). Examples of cell lines in which such studies have been carried out in include the mouse pituitary line AtT-20 (ATCC CCL 89), the rat pituitary growth hormone secreting lines GH3 (ATCC CCL 82.1), the insulin secreting .beta.TC lines derived from transgenic mice expressing SV40 T antigen (Efrat et al., 1988), radiation induced, rat islet cell tumor derived RIN lines (Gazdar, et al., 1980) and the rat adrenal pheochromocytoma cell line PC12 (ATCC CRL 1721). These cell lines maintain many of their endogenous functions, including synthesis of peptide hormones destined for the regulated secretory pathway. These cell lines also are transfectable, allowing expression of novel transgenes for studies of heterologous protein systems.
Three major areas have been studied using these heterologous systems. The first is the study of the sorting mechanism, whereby a given protein, destined for secretion, is targeted to the regulated secretory pathway or the default constitutive secretory pathway. The second relates to understanding the complex process of secretory protein maturation. This would include the specific steps of protein folding, disulfide bond formation, glycosylation, endoproteolytic processing and post-translational modifications of specific amino acids as well as understanding the enzymes involved in these processes. And the third relates to control of the regulated release of peptide hormones from secretory granules following physiological stimuli.
Neuroendocrine cell lines have been generated in which genes encoding specific peptide hormones have been stably inserted. These enzymes include insulin (Moore et al., 1983, Powell et al., 1988 and Gross et al., 1989), somatostatin (Sevarino et al., 1987), thyrotropin-releasing hormone (Sevarino et al., 1989), neuropeptide Y (Dickerson et al., 1987), insulin-like growth factor-1 (Schmidt and Moore, 1994), proopiomelanocortin (Thorne et al., 1989), glucagon (Drucker et al., 1986 and Rouille et al., 1994), pancreatic polypeptide (Takeuchi et al., 1991) and growth hormone (Moore and Kelly, 1985). In general, heterologous expression of these proteins has demonstrated faithful sorting to the regulated secretory pathway, as well as maturation of the proteins in the secretory granules. However, the expression levels of the heterologous proteins have generally been low when compared to normal endogenous expression of the same proteins in a homologous system.
Neuroendocrine cell lines expressing the enzymes involved in the processing of peptide hormones in secretory granules also have been generated. These include the endoproteases PC2 and PC3 (Ohagi et al., 1992, Benjannet et al., 1993, and Rouille et al., 1995) and peptidylglycine alpha-amidating monooxygenase (PAM) (Milgram et al., 1992 and Yun and Eipper, 1995). Overexpression of these processing enzymes has helped dissect their relative contributions to peptide hormone processing as well as their intracellular sites of action. These studies demonstrate the academic use of neuroendocrine cells in studying the regulated secretory pathway.
A series of papers over the last five years has addressed the possibility of production of heterologous peptide hormones in neuroendocrine cells. Three of these reports (Sambanis et al., 1990 and 1991, Grampp et al., 1992) use previously established AtT-20 lines expressing either insulin (Moore et al., 1983) or growth hormone (Moore and Kelly, 1985). The highest level of secretion of insulin under stimulated conditions was in the range of 35 to 144 microunits/million cells/hour (equivalent to 1 to 5 ng insulin/million cells/hr). Growth hormone secretion under stimulated conditions was 130 to 340 ng/million cells/hour. These levels of production are well below those reported in the literature for growth hormone production from other recombinant systems (Pavlakis and Hamer, 1983 and Heartlein et al., 1994). Another study dealing with protein production from a neuroendocrine cell makes use of an insulinoma line engineered to express prolactin (Chen et al., 1995). Absolute levels of production of prolactin on a per cell basis are not reported. A neuroendocrine cell-based system for either in vitro, biologically active peptide hormone production or for in vivo, cell-based delivery of biologically active peptide hormones has not been achieved in any of these earlier studies.
At least five important features must be addressed before a neuroendocrine cell-based system for protein production can be developed. The first feature is the absolute level of production of the polypeptide in question. A sufficiently high level of production to make either in vitro purification or in vivo efficacy must be achieved. As stated above, while many groups have reported expression of recombinant proteins in neuroendocrine lines, the proteins are produced at very low levels.
The second feature is the need for quantitative processing of the peptide to their biologically active forms. Neuroendocrine cell lines maintain variable levels of the enzymes responsible for peptide hormone processing and in many lines the enzyme levels may be insufficient to ensure sufficient processing. This is a critical parameter, especially as attempts are made to engineer high level production of specific peptide hormone transgenes.
The third feature is the need to maintain a dynamic response of the regulated secretory pathway. For both in vivo and in vitro use of a neuroendocrine cell-based system, the ability to quickly release high concentrations of the biologically active peptide by extracellular stimuli is important. In vivo modulation of peptide hormone release is required for titrating the biological efficacy of the cell-based delivery. In vitro modulation of peptide hormone release establishes efficient production of highly enriched fractions of starting material for subsequent purification.
The fourth feature is the ability to further engineer other functions into neuroendocrine cells other than just the high-level production of a given polypeptide. This further engineering could involve augmenting the cells capabilities such that any of the three previous points are improved or stabilized (i.e., increased protein levels, increased processing efficiencies or increased dynamic regulated secretory response).
The final engineering maneuver of significance is the ability to reduce or completely ablate the endogenous expression of an unwanted gene product. Reduction or ablation may result in an improved capability to produce, process or release the heterologous polypeptide. Such maneuvers also may confer advantages by removing unwanted or contaminating biological properties of the endogenous peptide hormone. Endogenous peptide production also might counteract the biological activities of the exogenous peptide hormone being produced, resulting in unwanted immunological reactions, reducing the capacity of the engineered lines to quantitatively produce the exogenously engineered protein or complicating purification of the exogenously produced protein. Because all of the existing neuroendocrine cell lines produce endogenous secreted proteins, these concerns are significant.
Thus, despite the benefits of developing a secretory cell line in which the protein synthetic machinery has been commandeered for the production of a heterologous polypeptide, there appear to be significant technical obstacles that are not addressed by the art. As a result, there currently exist no engineered cells that address all of these problems.